Advances in biotechnology have enabled the generation of plants which express recombinant proteins. Thus, plants can be engineered to overproduce a variety of polypeptides with desirable qualities. Such polypeptide can include enzymes which produce secondary metabolites, proteins with medicinal or pharmaceutical properties, and proteins which endow the plants with new traits, for example, resistance to diseases, pathogens, and environmental conditions.
Given the vulnerability of agricultural crops to damage by insects, and other pests and pathogens, the ability to provide additional protective means and agents is of considerable importance. Moreover, traditional breeding techniques have identified plant lines with Mendelian traits endowing resistance to pests and pathogens. Modern molecular biological techniques can now be applied to isolate the critical nucleic acids and proteins with these properties in order to enhance the resistance of more sensitive plants or to antagonize the pests and pathogens in a variety of scenarios.
The invention is based, in part, on the discovery of a novel mung bean nucleic acid which is expressed in a mung bean plant line that is resistant to insect attack, but not expressed in sensitive plant lines. The nucleic acid encodes a polypeptide which has insecticidal activity and which has similarity to thionin proteins. The sequence of the mung bean thionin nucleic acid (SEQ ID NO:1), designated as xe2x80x9cVrCRPxe2x80x9d, is shown below:
5xe2x80x2-ACCTCAACAATTCATCACTCATGGAGAGAAAAACTTTCAGCTTCTTGTT CTCGCTCCTTCTCGTCTTAGCCTCTGATGTGGCCGTAGAGAGAGGAGAGGCTAGA ACTTGTATGATAAAGAAAGAAGGGTGGGGAAAATGCTTAATTGACACCACCTGT GCACATTCGTGCAAGAACCGCGGTTACATAGGTGGAGATTGCAAAGGCATGACG CGCACCTGCTATTGCCTCGTCAACTGTTGAACCCTTTTCGAATATCATATCATCTT ATCACAAATAAATATAGCAGCATCACTGCTACTAGTACCGCCCTCCGCACCACG CCCT-3xe2x80x2
The initiator and terminator codons are underlined and in boldface. The sequence of the mung bean thionin polypeptide sequence (SEQ ID NO:2), designated as xe2x80x9cVrCRPxe2x80x9d, is shown below:
MERKTFSFLFSLLLVLASDVAVERGEARTCMIKKEGWGKCLIDTTCAHSCKNR GYIGGDCKGMTRTCYCLVNC
The invention is also based on the discovery the a polypeptide derived from VrCRP which has the VrCRP signal sequence removed is biologically active as an insecticide and fungicide. This polypeptide is encoded by the nucleic acid sequence (SEQ ID NO:3) below:
GAGAGAGGAGAGGCTAGAACTTGTATGATAAAGAAAGAAGGGTGGGGAA AATGCTTAATTGACACCACCTGTGCACATTCGTGCAAGAACCGCGGTTACATAGGT GGAGATTGCAAAGGCATGACGCGCACCTGCTATTGCCTCGTCAACTGTTGA
The polypeptide sequence (SEQ ID NO:4) of this form of VrCRP lacking the signal sequence is shown below:
ERGEARTCMIKKEGWGKCLIDTTCAHSCKNRGYIGGDCKGMTRTCYCLVNC
Accordingly, in one aspect, the invention features isolated nucleic acid sequences which include a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO:1. The nucleic acids of the invention can further include nucleic acids which hybridize under stringent conditions to SEQ ID NO:1, as well as nucleic acids which are at least 50% identical, e.g., at least 60%, 70%, 80%, 90%, or 95% identical, to SEQ ID NO:1. Such nucleic acid sequences can encode a polypeptide which inhibits translation of messenger RNAs in a wheat germ extract, a polypeptide which has insecticidal activity, e.g., insecticidal activity against bruchids such as Callosobruchus chinensis, Callosobruchus maculates, and Zabrotes subfasciatu, or a polypeptide which has anti-fungal activity, e.g., against Rhizoctonia solani. 
In another aspect, the invention features polypeptides comprising the amino acid sequence of SEQ ID NO:2. Featured polypeptides also include polypeptides which are at least 50% identical, e.g., at least 60%, 70%, 80%, 90%, or 95% identical, to SEQ ID NO:2. Such polypeptides can have at least one, two, three, four, five, eight, ten, twelve, or twenty conservative amino acids substitutions. The polypeptides can inhibit translation of messenger RNAs in a wheat germ extract, can have insecticidal activity, e.g., insecticidal activity against bruchids such as Callosobruchus chinensis, Callosobruchus maculates, and Zabrotes subfasciatus, or can have anti-fungal activity, e.g., against Rhizoctonia solani. Also encompassed by the invention are nucleic acid sequences encoding such polypeptides.
The featured polypeptides can be recombinant and/or purified. For example, they can be overexpressed in a variety of host cells, such as E. coli, Sf9 insect cells, plant cells and mammalian tissue culture cells using overexpression vectors known in the art. Lysates are made from the host cells, e.g., after overexpression is induced if induction is required. The polypeptides are purified from the lysate. Alternatively, the polypeptides are secreted, by the inclusion nucleic acid sequences encoding the signal peptide or a heterologous signal peptide. In another example, the featured polypeptides are encoded by a transgene and overproduced in a plant or a plant tissue. The plant is harvested and the polypeptides purified from the plant.
The purified and/or recombinant polypeptides can be formulated a composition. The composition can include an agriculturally acceptable carrier, e.g., one described below. The composition can contain the polypeptide at a concentration of about 0.005% to 10%, or about 0.01% to 1%, or about 0.1% to 0.5% by weight of composition. The composition can further include a cyclopeptide alkaloid, e.g., vignatic acid A and vignatic acid B, e.g., a cyclopeptide alkaloid with insecticidal properties. The composition can also include other desirable compounds, e.g., protease inhibitors, endotoxins, and the like. The contemplated compositions can have insecticidal activity, e.g., against bruchids such as Callosobruchus chinensis, Callosobruchus maculates, and Zabrotes subfasciatu, and/or anti-fungal activity, e.g., against Rhizoctonia solani. The compositions can be applied to plants and their environs by methods described below.
The nucleic acids of the invention can also include a heterologous promoter such that the promoter is operably linked to a coding genomic nucleic acid or a cDNA. The promoter can direct transcription of the nucleic acid in wounded or pathogen infected cells. The promoter can be induced by a signalling molecule, e.g., methyl jasmonate, salicylic acid, ethylene, absiscic acid, gibberillins, HgCl2, and H2O2. The invention also features transformed cells which contain such nucleic acids, i.e., an aforementioned nucleic acid operably linked to a heterologous promoter. Also included are transgenic plants whose genomic DNA includes such nucleic acids, as are transgenic seeds from such plants.
A xe2x80x9cpurified polypeptidexe2x80x9d, as used herein, refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide can constitutes at least 10, 20, 50 70, 80 or 95% by dry weight of the purified preparation.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is detemined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be perfonned with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
As used herein, the term xe2x80x9ctransgenexe2x80x9d means a nucleic acid sequence (encoding, e.g., one or more subject thionin polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic plant or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic plant or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the plant""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic plantxe2x80x9d is any plant in which one or more, or all, of the cells of the plant includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by T-DNA mediated transfer, electroporation, or protoplast transformation. The transgene may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as a leaf, a root, or a stem.
As used herein, the term xe2x80x9chybridizes under stringent conditionsxe2x80x9d refers to conditions for hybridization in 6xc3x97sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97SSC, 0.1% SDS at 50-65xc2x0 C.
A xe2x80x9cheterologous promoterxe2x80x9d, when operably linked to a nucleic acid sequence, refers to a promoter which is not naturally associated with the nucleic acid sequence.
As used herein, an agent with xe2x80x9cinsecticidal activityxe2x80x9d is an agent which when tested by the following assay has measurable insect-killing activity. In the assay, the agent is combined with mung bean flour produced from an insect-sensitive mung bean strain and packed as an artificial bean. Insect eggs are placed on the artificial bean. The timing and number of hatched insects are measured. An agent with xe2x80x9cinsecticidal activityxe2x80x9d demonstrates delayed hatched, e.g., a delay of about 2, 4, 5, 7, 10, or 14 days. Alternatively, the agent can prevent hatching, e.g., only about 80%, 60%, 40%, 30%, 10%, or 0% of the eggs hatch after 14 days.
As used herein, an agent with xe2x80x9cfungicidal activityxe2x80x9d is an agent which when tested in the following assay produces a measurable zone of growth inhibition. The agent, in an acceptable solvent, is soaked in a sterile filter disc which is placed on an agar plate top spread with a fungus, e.g., Rhizoctonia solani. The plates are incubated, e.g., for 36-48 hours, and then zone of growth inhibition around each filter disc is measured. An agent with xe2x80x9canti-fungal activityxe2x80x9d produces a zone of inhibition of about 1, 2, 3, 4, 5 mm or greater after 36 hours of fungus growth.
As used herein, an agent which xe2x80x9cinhibits messenger RNA translation in a wheat germ extractxe2x80x9d is an agent which when present in an in vitro translation reaction obtained by combining a messenger RNA with a wheat germ extract, e.g., a commercial wheat germ extract, prevents incorporation of [35S] methionine into an acid insoluble fraction by at least 30%, e.g., at least about, 50%, 75%, or 100%.
The discovery of a polypeptide thionin from insect resistant mung beans with insecticidal properties and its encoding nucleic acid sequence has a variety of commercial and agriculture benefits. The polypeptide can be used in a composition, e.g., a composition which includes agriculturally acceptable carriers, or other agents, to protect plants from insects, and other pests and pathogens. The composition can be formulated and applied by methods described herein in order to protect a plant. In another aspect, the nucleic acid can be used to generate a transgenic plant which expresses the thionin to thereby protect the plant. By conferring resistance to damage by insects, these strategies are of considerable economic benefit.
Other features, objects, and advantages of the invention will be apparent from the description and from the claims.